Immuncassays can be used to detect the presence of unwanted agents in a sample of fluid, for example the presence of bacteria in a bodily fluid or a pollutant in water. They can be used also to test for the presence of antibodies in body fluids, as an indication of the presence of an infectious agent, a cancer cell or an autoimmune disease.
A variety of test assay components have been developed for use in immunoassavs, and some of these components exploit the capillary flow of fluid through a porous medium. Assays utilizing this phenomenon have occasionally been somewhat improperly termed "chromatographic assays", and some involve but a one-step procedure. For example, U.S. Pat. No. 4,956,302, issued Sep. 11, 1990 to Gordon et al., describes an assay device including a chromatographic medium having a reaction site at which a specific binding reagent is immobilized, a sample application well located adjacent to the chromatographic medium and offset upstream from the reaction size, and a liquid absorption blotter offset downstream from the reaction site. A fluid sample is applied to the application well and is transported through the chromatographic medium, through the reaction site, and to the blotter. The reaction site is then contacted with a labeled, specific binding material, is washed to remove unbound species, and the label is detected.
U.K. Patent Application Number 8809867, published Nov. 9, 1988 under Publication Number 2204398A, describes a nitrocellulose medium including a sample application area, non-immobilized, labeled binding reagent specific for a first analyte epitope located downstream therefrom, and immobilized, non-labeled binding reagent specific for a second analyte epitope located further downstream. The analyte is provided in a fluid which carries the analyte to the labeled reagent and which carries the analyte and labeled reagent together to the immobilized, non-labeled reagent. After the assay, observation of label at the zone of immobilization is indicative of the presence of analyte in the sample.
in each of the above-described devices, the chromatographic medium provides the function of filtering the fluid sample of unwanted particulates, and of slowing the flow of fluid to allow specific bindlng to occur and to efficiently carry non-immobilized reagents to sites where other reagents are immobilized. However, this slow flow contributes to non-specific binding as well, and one must wait for a period of time on the order of minutes for a test result.
Filtration of large quantities of serum and blood has also been carried out using porous media such as nitrocellulose. However, a significant pressure differential must be applied across a nitrocellulose filter to achieve such filtration.
Porous media have been used to form so-called "prefilters" in test assay apparatus, these prefilters being used to spread a sample from a localized application area over a larger test assay surface area. U.S. Pat. No. 4,912,034, issued Mar. 27, 1990 to Kalra et al. describes such a prefilter. These prefilters are typically constructed of glass fibers, and provide rapid wetting of a test assay surface, but provide poor filtration, as selection of a prefilter having a pore size providing adequate filtration generally slows the wetting process significantly. That is, to apply a sample using a prior art prefilter, good filtration and rapid wetting of an assay surface are generally mutually exclusive. Additionally, glass fibers have very large void (retention) volumes, hence they absorb significant quantities of sample. Therefore, if only a small sample is available, it must be diluted significantly if the assay surface is to be adequately wetted. Then, to boost sensitivity, an enzymatic tag is typically employed.
Although many such assays represent extraordinary improvements over one prior methodology for determining the presence of an agent in a sample, the goal remains to develop immunoassays that are rapid and sensitive. It is a drawback of almost every immunoassay presently in use that the assay either is too slow (5-30 minutes) or, if faster, is not sufficiently sensitive (or both). Where the detection of disease is concerned, it is unacceptable to compromise on sensitivity, and accordingly those assays that are among the most sensitive tend to be cumbersome and slow.